The first goal of this project is to establish the physiological significance of myosin polymorphism in mammalian skeletal muscle. The basic hypothesis to be tested is that each myosin isozyme has unique force-velocity and energetic properties. Rather than generalizing the properties of fast-twitch or slow-twitch mammalian fibers, this project seeks to establish the quantitative contractile and biochemical differences among subpopulations of fast- and slow-twitch fibers, and even among subpopulations of fibers that have been nominally lumped into the same histochemical type. The second goal of the project is to begin to dissect the functions of the myosin subunits. The project will be able to separate functional contributions of myosin light chains vs myosin heavy chains by creating altered combinations of heavy and light chains within permeabilized muscle fibers. This project will also involve the construction of a number of light chain mutants, which will be expressed in bacteria and reconstituted into permeabilized fibers. The scope of the investigations will include (1) properties of permeabilized rabbit fiber segments with their naturally occurring complement of contractile proteins; (2) permeabilized fibers in which exchange of the myosin light chains with either naturally occurring light chains or bacterially synthesized mutant light chains has been performed; and (3) purified myosin isozymes assessed in an in vitro assay. Contractile and biochemical measurements will include force per cross-sectional area, force-velocity relationship, pCca-force relationship, isometric actomyosin ATPase activity, the rate of isometric redevelopment of force and cross-bridge kinetics determined using "caged" ATP. This project seeks to uncover the significance of the range of myosin isozyme distributions found in mammalian skeletal muscle, and to delineate the functional contributions of each of the myosin subunits. The problem takes on greater significance when one considers that the isozyme composition of skeletal muscle is not necessarily static. Mammalian skeletal muscle cells show activity-dependent changes in expression of the various contractile proteins isoforms as well as levels of metabolic enzymes. Plasticity is relevant not only to normal adaptation to altered activity but also to adaptation that occurs in neuromuscular disease states.